Simplified Equation for Photosynthesis:
    6 CO₂ + 6 H₂O ® C₆H₁₂O₆ + 6O₂ Endergonic reaction: delta G = +686 kcal mol^-1

Basic Dark Reactions Process:

C₃ Photosynthetic Carbon Reduction Cycle (PCR cycle).
Occurs in virtually all autotrophic organisms - even in those with auxiliary pathways or cycles. Also termed Calvin cycle (Melvin Calvin and co-workers worked it out in early 1950s).

It is the way to reduce CO₂ to carbohydrates without losing organic-C in the process.
There are other CO₂ fixation (carboxylation) processes, eg PEP carboxylase, but in all cases CO₂ has to be released again (decarboxylation) before entering carbohydrates.

Other Dark Reactions Processes:

 C₂ Photorespiratory Carbon Oxidation Cycle (PCO cycle) and how it is intimately linked thru Rubisco to the PCR cycle.

Concept of CO₂ concentrating Mechanisms (CCMs). C₄ cycle, CAM, HCO^-₃ pumps.

C₄-acid cycle ( C₄ species) and how this cycle overcomes the operation of the PCO cycle, and thus reduces PHOTORESPIRATION.

Crassulacean Acid Metabolism (CAM) which is a variation on the C₄ cycle to conserve H₂O (and CO₂).

Algal and Cyanobacterial CO₂/HCO^-₃ pumps which concentrate CO₂ inside the cell. Improves CO₂ uptake at low [CO₂] and reduces photorespiration.

Regulation of Sucrose and Starch Metabolism. Sucrose for export from the leaf, starch for storage in the chloroplast.

• Requires ATP and NADPH from the light reactions to function

• From PGA in the PCR cycle can form amino acids, fatty acids and nucleic acids

• From triose-P in the PCR cycle can synthesize carbohydrates (sucrose and starch, and cellulose)

• Regenerates ribulose bisphosphate  (RuBP) to continue the cycle.

Can be subdivided into three major stages (Fig 8.2):
                1. Carboxylation Stage
                2. Reduction StageRuBP
                3. Regeneration Stage

    All occur in stroma where ATP and NADPH are located after their synthesis in light reactions
    Requires 13 enzymes and reactions
    Forms two C₃ acids: P-glycerate and bisP-glycerate (old name diP-glycerate) - PGA and BPGA
    Rest of compounds are sugar-P or sugar bisP of C₃, C₄ , C₅, C₆ and C₇ carbons.

                Elucidated by use of radioactive isotopes - mainly ¹⁴CO₂.
                How?                             What is the most common C isotope?

First Three Reactions of PCR cycle: Table 8.1 and Fig 8.4.

6 RuBP (30 C) will react with 6CO₂  (6 C) to give 12 x PGA (36 C). Of these 36 carbons, 30 are recycled to RuBP (6 x RuBP = 30 C) and 6 can be drained off as 2 x GAP (6 C) to make glucose (6 C).

                   How can C₃ (GAP) be converted to C₅ (RuBP)?

                    C₃  +  C₃   =   C₆                                    C₆  +  C₃   =   C₄   +   C₅
                    C₄  +  C₃   =   C₇                                    C₇  +  C₃    =   C₅  +   C₅

Stochiometry of PCR Cycle:

1 mol hexose sugar (2 x GAP) synthesized needs 6 mol of CO₂.  This requres 18 mol of ATP and 12 mol NADPH.  Thus the PCR cycle needs a ratio of  3 ATP : 2 NADPH.

Thermodynamic efficiency:

• 1 mol photons at 680 nm wavelength = 175 kJ
• A minimum of  8 photons is needed per 1 CO₂ fixed = 1,400 kJ
• 6 CO₂ are needed per hexose = 8,400 kJ.
• But 1 mol hexose yields only 2804 kJ.

In nature (agriculture) actual efficiency of solar energy conversion to biomass (yield) is much less from 0.5 to 3%.

Regulation of PCR Cycle:  Figs  8.5 and 8.6

Coarse control: Amount of enzyme protein (esp. Rubisco) determined by mRNA expression and protein degradation rate.

Fine Control:    Three impotant regulatory enzymes in the PCR cycle:

Rubisco,         Fructose bisphosphatase (FBPase),         P-ribulokinase.

                                             What does LIGHT do?

• H⁺ pumping from stroma to lumen - stromal pH rises to 8.2 (pH optimum for these enzymes)
• Mg²⁺ exchanged for H⁺, so stromal Mg²⁺ rises and it activates (cofactor) Rubisco. Rubisco needs activation with CO₂ and Mg²⁺ before it can catalyze carboxylation reaction.
• Rubisco activase (enzyme) activates Rubisco in light.
• Ferredoxin - thioredoxin system (from light reactions) activates FBPase and P-ribulokinase by reducing cysteine residues from -S-S-  to  -SH   HS-.  Fig 8.5.

• Why is the solar energy conversion efficiency usually less than 3% in agricultural and natural systems?
• What is the theoretical maximum energy conversion efficiency for photosynthesis?
•  What processes and factors reduce it from its theoretical maximum?

• World’s most abundant enzyme (protein)
• Catalyzes » 200 billion tons CO₂ fixed y^-1.
• Is about 40% of leaf protein (4 mM active sites in stroma).
• The reaction by which inorganic carbon enters biosphere organic-C pool. (Most other carboxylation reactions require loss of C for each C fixed).

    Large, sluggish enzyme (molecular mass 560 kD).
    L₈S₈. Eight active sites (one on each large subunit).
    Large subunit encoded in chloroplast, small by nuclear genome.
    Very negative delta G (» 50 kJ) so reaction goes virtually to completion (ie to PGA).
    K_m(CO₂) ca 10mM in C₃ plants but ca 30mM  in C₄ plants.
    Note - At 25°C dissolved air-CO₂ is about 10 mM but only 2-4 in C₃ chloroplast
    So, enzyme working at below K_m in C₃ plants, not at V_max.

    O₂ inhibits Rubisco.  O₂  competes with with CO₂. Note that air contains both O₂ (21%) and CO₂ (0.37%).
    O₂ with its similar structure to CO₂ can also react with RuBP in an OXYGENASE reaction (K_m(O₂) ca 250 mM).

        Initiated by Rubisco (as is the PCR cycle)

                                                                     Rubisco
                                           RuBP    +    O₂   ®    P-glycolate    +       PGA    +    H⁺
                                         P-C₅-P                                 C₂-P                    C₃-P
                                                                                         ¯                          ¯
                                                                                   PCO cycle             PCR cycle

        Flow of carbon in C₃ leaf is determined by the balance between PCR and PCO cycles. Rubisco initiates both.
        PCR = net C gain.  PCO = net C loss (Fig 8.8).

See Fig 8.7 & Table 8.2 for the PCO cycle

• Only occurs in light.  Respiration occurs in the dark and to some extent in the light.

• It has net consumption of ATP (uses up energy).
• Its substrate is P-glycolate (C₂-P), not glucose.
• It is [O₂]-limited at air (21%) oxygen concentrations, and inhibited by CO₂.
• Releases CO₂ from mitochondria (both processes do this).
• Uses three organelles for the PCO cycle: chloroplast, peroxisome, mitochondrion.
• Initiated by Rubisco (like PCR cycle)

Cyanobacterial and Microalgal CCMs (P 156)

• Microalgae grown at 5% CO₂ are photosynthetically like C₃ plants (photorespiration) but when switched to low CO₂ (air) within a few hours their affinity for CO₂ increases - they have a low apparent K_m (CO₂) and O₂ has no effect on photosynthesis. Why?
• Low [CO₂] induces the ability to pump HCO₃^- into cell and produce high internal CO₂ concentrations.
• High [CO₂] in carboxysomes of cyanobacteria (where Rubisco and carbonic anhydrase are located) or in chloroplasts (pyrenoids) of microalgae outcompetes O₂ for Rubisco so the PCO cycle cannot operate.
• The CCM enables these organisms to grow at low external [CO₂] and not lose CO₂ through the PCO cycle.

• Enhances supply of CO₂ to Rubisco. Overcomes (outcompetes) the O₂ inhibition of rubisco and photosynthesis, and thus prevents photorespiratory CO₂ loss.
• A CCM or CO₂ pump. Increases [CO₂] to over 70 mM around Rubisco. (What is air-equivalent value?) "Supercharger for PCR cycle."
• Occurs more in tropical species. Most crops are C₃, but maize, sugarcane, sorghum are C₄ and highly productive.
• Most advantageous in warm, drier, high light areas (improves water use efficiency).
• PEP carboxylase (PEPC) is the initial carboxylase. What's the advantage of PEPC?
• PEP  +  HCO₃^-  =   OAA  +  Pi
• Forms oxaloacetic acid (OAA), which is a C₄ dicarboxylic acid, as the first product of CO₂ fixation. Hence the term "C₄" photosynthesis.
• Needs Kranz leaf anatomy.

Overall C₄ Scheme.  Fig 8.10.
Carboxylation, decarboxylation, regeneration.  HCO₃^- fixed by PEPC in mesophyll. Forms C₄ acid for transport to the bundlesheath. C₄ acid is decarboxylated to concentrate CO₂ in BSC for refixation by Rubisco into the PCR cycle. The substrate for PEPC (i.e PEP) is regenerated.

Three Types of C₄ Photosynthesis:
            They are based on three different decarboxylases in bundlesheath cells:

NADP - malic enzyme (NADP-ME) C₄ species  -  a bundlesheath cell chloroplastic decarboxylase
NAD - malic enzyme (NAD-ME) C₄ species  - a bundlesheath cell mitochondrial decarboxylase
PEP carboxykinase (PEPCK) C₄ species  - a bundlesheath cell cytosolic decarboxylase.

• All separate the initial fixation CO₂ by PEPC (in the mesophyll cytoplasm) from the decarboxylase and subsequent refixation by Rubisco (in the bundlesheath cells). This prevents futile cycling of CO₂.

• All concentrate CO₂ in the bundlesheath cells.
• Rubisco and the PCR cycle are ONLY in bundlesheath cell chloroplasts NOT in mesophyll cell chloroplasts. Why?
• All require transport of C₄ acid (malate or aspartate) into bundlesheath cells  for decarboxylation and refixation of released CO₂ into PCR cycle. High [CO₂] overcomes the O₂ effects on Rubisco.

• Pyruvate or alanine return to mesophyll cell for conversion to PEP (phosphoenolpyuvate) - one of the PEPC substrates (other is bicarbonate ion).

                                                   NADP-ME                   NAD-ME                   PEPCK

    ATP Required
                        C₄ Cycle                   2                                        2                              3
                       PCR Cycle                 3                                        3                              3
                       Total ATP                  5                                        5                              6

    NADPH Required
                        C₄ Cycle                     1                                        0                               0
                       PCR Cycle                  1                                        2                               2
                       Total NADPH             2                                        2                               2

For C₄ mechanisms see Figs 8.10 and 8.11

CAM Photosynthesis  Fig 8.12.

• Major role is water and CO₂ conservation system.  (WUE = water use efficiency)

• Similar biochemistry to C₄  But PEPC activity separated from Rubisco and decarboxylase by TIME not SPACE.
• Thus stomates open at NIGHT:

•  PEP comes from carbohydrates that were produced and stored during the previous day
•  HCO₃^-is produced as CO₂ from the air dissolves in the cell water.
•  Malic acid is stored during the night in the large cell vacuole.
• Thus cell acidity increases at night.
• The stomates close during the DAY.  WHY?
• Malate is exported from vacuoles.
• Malate or OAA are decarboxylated to release CO₂ in leaf (1-2% CO₂). Why can’t CO₂ escape to air?
• Because it is light (day) the light reactions can make ATP and NADPH. The PCR cycle runs and CO₂ is fixed by Rubisco and eventually is assimilated into sugars and starch (storage).
• Large diel fluctuations in leaf acidity (first seen in cacti hence the acronym CAM for Crassulacean Acid Metabolism). High [malic acid] at end of night, low at end of day.

    Why doesn’t PEPC also fix the CO₂ during the day - and compete with Rubisco for inorganic carbon?

• PEPC enzyme is subject to covalent modification (reversible phosphorylation) which alters its sensitivity to allosteric inhibition (feedback inhibition) by malate.
• Low inhibition at night when [malate] is low; high inhibition at day when [malate] high.
• Thus PEPC is active at night; not during the day.

Several variations on CAM theme:

• Obligate CAM (can only do CAM);
• Facultative CAM - C₃ photosynthesis in leaves when plenty of water, switch to CAM under adverse conditions of water, salt, temperature, photoperiod or CO₂
• CAM idling (stomates closed day and night when water is unavailable, leaf refixes, ie. recycles respiratory carbon dioxide during the day).

• What are the major anatomical differences between C₃ and C₄ leaves?
• What is (are) the biochemical difference(s) between C₃ and C₄ photosynthesis?
• Do both use the PCR cycle?
• Do both operate the PCO cycle?
• In a single phrase, what does the  C₄ cycle ultimately accomplish?

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